1. Field of the Invention
The present invention relates to uronium and immonium salts and their use in effecting the acylation step in amide formation, especially during peptide synthesis.
2. Description of the Prior Art
Polypeptides are useful as medicaments. In recent years, peptides have been found to be useful in combating various diseases, such as cancer, diabetes, plant toxins and the like. Additionally, peptides have shown specific activity as growth promoters, suppressants, antibodies, insecticides, contraceptives, anti-hypertensives, sleep-inducers, anti-depressants, analgesics, etc. The list is long and varied.
As more and more polypeptides become of medicinal importance, there is an increasing incentive to improve the methods by which they may be synthesized. Currently, syntheses of peptides are in solution by classical or various repetitive methods. Alternatively, peptides may be prepared on a solid support (Merrifield method). These are all popular techniques in synthesizing peptides from the coupling of two or more amino acids, in synthesizing larger peptides from the coupling of amino acids with smaller peptides or in the coupling of smaller peptides. Solution methods have the advantage of being easily monitored, allowing purification of intermediates, if necessary, at any stage. A major drawback, however, is the relative slow pace of synthesis, with each step being carried out manually.
The major advantage of the Merrifield method is its easy automation so that unattended, computer-controlled machine synthesis is possible. Unfortunately, the method suffers from an inherent deficiency due to the insoluble nature of the support on which the synthesis proceeds. Unless each acylation step occurs with approximately 100% efficiency, mixtures will inevitably be built up on the polymer. The longer the chain, the greater will be the contamination by undesired side reactions. Side products produced in such reactions remain to contaminate the desired product when it is removed from the polymeric matrix at the end of the cycle. These current techniques are not useful in preparing peptides of greater than 30-40 residues; separation of side products from the desired product becomes increasingly difficult when larger peptides are synthesized.
For very long segments (60 or more amino acids), therefore current methods are not satisfactory. Often, mixtures are obtained of such forbidding complexity that it may be difficult or impossible to isolate the desired peptide.
The problems enumerated hereinabove may be eliminated if the proper derivatives of the underlying amino acids and/or the proper conditions for the coupling reaction could be found. Protecting groups, such as t-butyloxycarbonyl (t-Boc) or N-xcex1-(9-fluorenylmethyl)oxycarbonyl (Fmoc), have been used to minimize side reactions. But, additionally, other aspects of the coupling reaction must also be taken into consideration, such as the peptide coupling additive to be used in the coupling reaction.
Additives generally inhibit side reactions and reduce racemization. Heretofore, the most common peptide coupling additive used during peptide coupling for both solutions and solid phase synthesis is 1-hydroxybenzotriazole (HOBt). This reagent has been used either in combination with a carbodimide or other coupling agent or built into a stand-alone reagent, such as 1-benzotriazolyoxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) or an analogous uronium salt. HOBt is applicable to both stepwise and segment condensations. However, many cases have been encountered in which HOBt is ineffective, possibly because of stearic effects, or low basicity of the amino component. Especially problematic are segment coupling at amino acid units other than glycine or proline, since the problem of racemization may be severe. The related N-hydroxybenzotriazinone (HOOBt) may provide better protection against racemization, but it is rarely used due to competing side reactions involving ring openings.
Other reagents for facilitating peptide coupling have also been described. For example, in Tetrahedron Letters, 1994, 35, 2279-2282, Carpino, et al. disclose that 1-hydroxy-7-azabenzotriazole and its corresponding uronium salts, designated HAPyU and AOP were effective in promoting peptide coupling and avoiding racemization in a model solid-phase peptide segment coupling process. In addition, Carpino, et al. disclose in J. Org. Chem., 1994, 59, 695-698 that azabenzotriazolyluronium salts, e.g., designated as HBTU, HATU, HBPyU, HAPyU, HBMDU and HAMDU, are useful in peptide synthesis. Other publications such as Ehrlich, et al., disclose that the uronium salts, designated as HAPyU and TAPipU were useful for promoting peptide cyclization with a minimum of racemization.
U.S. Pat. No. 5,644,029 to Carpino discloses among other things, the use of compounds of the following formula in promoting peptide coupling: 
and N-oxides thereof and salts thereof wherein
R1 and R2 taken together with the carbon atoms to which they are attached form a heteroaryl ring wherein said heteroaryl ring is an oxygen, sulfur or nitrogen containing heteroaromatic containing from 3 and up to a total of 13 ring carbon atoms, said heteroaryl may be unsubstituted or substituted with lower alkyl or an electron-donating group;
Y is O, NR4, CR4R5;
R5 is independently hydrogen or lower alkyl;
X is CR6R7 or NR6;
R6 and R7 are independently hydrogen or lower alkyl; or R6 and R7 taken together form an oxo group or when n=0, R4 and R6 taken together may form a bond between the nitrogen or carbon atom of Y and the nitrogen or carbon atom of X;
Q is (CR8R9) or (NR8);
when n is 1, R4 and R8 taken together may form a bond between the ring carbon or nitrogen atom of Q and the ring carbon or nitrogen atom of R8;
n is 0, 1 or 2;
R3 is hydrogen, lower alkyl carbonyl, aryl carbonyl, lower aryl alkyl carbonyl, 
a positively charged electron withdrawing group, SO2R14, or 
R14 is lower alkyl, aryl or lower arylalkyl; q is 0-3;
R8 and R9 are independently hydrogen or lower alkyl or R7 and R8 taken together with the carbon to which they are attached form an aryl ring, AA1 is an amino acid and BLK is an amino protecting group, and m is 0 or 1.
It teaches that the compounds are prepared by reacting compounds of the formula: 
with R3L in the presence of a base under substitution reaction conditions, in which R1, R2, Y, Q, n, X, and R3 are as defined hereinabove and L is a leaving group, such as halide.
At the time of the publications of the aforementioned articles as well as of the time of the filing of the aforementioned patent, it was believed that all of the compounds described therein had the formula shown hereinabove wherein the R3 was bonded to the oxygen atom (the O-isomer). This belief was based upon the structure of the corresponding phosphonium derivatives, such as benzotriazol-1-yl-N-oxy-tris(dimethylamino) phosphonium hexafluorophosphite (BOP) 
and benzotriazol-1-yl-N-oxy-tris-(pyrrolidino)-phosphonium hexafluorophosphate 
in which the oxygen atom was bonded to the cationic phosphonium group. Based on these structures, when the uronium salt derivatives of hydroxybenzotriazole were first described it was assumed, by the scientific community by analogy, to have the structure hereinbelow: 
wherein the positively charged uronium ion was bonded to the oxygen atom.
In addition, when other coupling reagents, such as HATU were described, by analogy to the structures assigned to the hydroxybenzotriazole derivatives, it was also assumed that HATU and its derivatives also had the structure: 
In fact, based on the same assumptions, it was believed that the uronium salts in general, for example, described hereinabove in the aforementioned publications had similar structures wherein the oxygen atom was bonded to the positively charged cation. Because such structures were believed to be the O-isomers, x-ray crystallography of these new O-isomers was not performed.
However, finally when x-ray crystallography was finally taken of the structures of HBTU and HATU, it was surprisingly learned that the assumption was incorrect with respect to the uronium salt derivatives. More specifically, it was later learned from X-ray crystallographic analysis that the structure assigned to HATU and HBTU were not the structures indicated hereinabove. More specifically, the positively charged moiety is not attached to the oxygen atom, but instead is substituted on the nitrogen atom of the triazole, having the structure shown hereinbelow: 
wherein Xxe2x95x90N or CH. In the triazole derivatives depicted hereinabove, this phenomenon wherein the positively charged group was on the nitrogen atom only appeared to occur when R3 in the structure above was an electron withdrawing group which contained a positively charged nitrogen atom.
However, the situation was even more complex. The present inventors noticed that when the 1-hydroxy-4-methyl-7-azabenzotriazole was reacted with 2-chloro-1,1,3,3-bis tetramethylene uronium hexafluorophosphate in the presence of a weak base, an interesting phenomenon occurred. Sometimes, they obtained the product 
But, other times, they obtained the product, 
and sometimes they obtained a mixture of the two. Even though the present inventors had conducted the reaction using the same reagents, the products obtained were not always the same. Until recently, the present inventors could not explain these different results with the 4-methyl derivatives, and the inventors did not understand or know how to make the O or the N-isomer of the 4-methyl derivative with any consistency.
Thus, it was concluded that in general, with respect to the triazole derivatives or triazole like derivatives, when R3 is a positively charged electron withdrawing group containing a positively charged nitrogen atom such as an amino cation or an uronium group, the product prepared in accordance with the methodology described hereinabove was not the O-isomer (i.e., the product in which the R3 group is attached to the oxygen atom), but rather the N-isomer (i.e., the product in which the R3 group is attached to the nitrogen atom). Thus, to date, when R3 is an uronium cation or immonium cation, the N-isomer has been prepared, but the corresponding O-isomer has not been prepared.
Based upon this revelation, there were concerted efforts in the scientific community to make the elusive O isomer for HOAT and HOBT immonium and uronium type coupling agents. For instance, Li and Xu alleged that they have found a means of making O isomers of various HOBT and HOAT immonium type coupling reagents, which were prepared in situ and which were useful in peptide coupling. For example, it was alleged but not confirmed by Li and Xu in Tetrahedron 56, 4437-4445 (2000) and in Tetrahedron Letters, 41, 721-724 (2000), that the reaction of the hydroxy triazole in the presence of SbCl6, would produce the O-isomer of HOBt or HOAT based immonium type reagents, such as those shown hereinbelow: 
However, the x-ray diffraction determined that they did not make the analogous O isomer derivative shown hereinbelow: 
but rather the N-isomer 
Moreover, when they took the x-ray analysis of another one of the compounds, namely BDMP, which they originally believed was the O-isomer, as drawn hereinabove, they also found that they did not make the O-isomer depicted hereinabove, but rather the corresponding N-isomer. See, Li and Xu, J. Chem. Soc. Perkin Trans., 2, 113-120 (2001). To date, they have not confirmed the structures of the other O-isomer products, which they have proposed.
Thus, to date, no one has actually prepared the O-isomers of the uronium salts and immonium salts of the HOAt or HOBt compounds.
However, the present inventors have found a means of synthesizing the O-isomers and have shown that the O-isomer is also useful for peptide coupling.
Accordingly, the present invention is directed to a compound of the formula: 
wherein
R3 is a positively charged electron withdrawing group having the formula: 
R10, R11, R12, R13, R14, R15, R16 are independently hydrogen or lower alkyl which may be unsubstituted or substituted with an electron withdrawing or electron donating group, or R10 and R12 taken together with the nitrogen atom to which they are attached and the carbon atom attached to the nitrogen atoms form a 5 or 6 membered nitrogen containing heterocyclic containing 3 or 4 ring carbon atoms, respectively or R10and R11 taken together with the nitrogen atom to which they are attached or R12 and R13 taken together with the nitrogen atom to which they are attached form a 5 or 6-membered heterocyclic ring containing up to 5 ring carbon atoms respectively or R14 and R15 taken together with the nitrogen atom to which they are attached form a 5 or 6-membered heterocyclic ring containing 4 or 5 ring carbon atoms, respectively or R14 taken together with the nitrogen to which it is attached and R16 taken together with the carbon atoms attached thereto form a 5 or 6 membered nitrogen containing heterocyclic ring, containing 4 or 5 ring carbon atoms, respectively;
E is N or CR;
R is hydrogen or lower alkyl;
SB is an anion;
Ra and Rb are independently hydrogen, lower alkyl, an electron withdrawing group or electron donating group or Ra and Rb taken together with the carbon atoms to which they are attached form a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group, which cycloalkyl, heterocyclic, aryl and cycloalkyl groups are unsubstituted or substituted by lower alkyl, an electron withdrawing group or an electron donating group.
In a preferred embodiment, the cation portion of the salt of Formula I has the formula: 
or the N-oxides thereof or the salts thereof wherein
A is an aryl group containing 6-14 ring carbon atoms and up to a total of 20 carbon atoms or a heteroaryl ring, where said heteroaryl ring is an oxygen, sulfur or nitrogen containing heteroaromatic having from 5 and up to a total of 14 ring atoms and from 3 up to a total of 13 ring carbon atoms and up to a total of 20 carbon atoms, said heteroaryl and aryl groups may be unsubsituted or substituted with an electron donating or electron withdrawing groups or lower alkyl;
R3 is a positively charged electron withdrawing group having the formula: 
R10, R11, R12, R13, R14, R15, R16 are independently hydrogen or lower alkyl which may be unsubstituted or substituted with an electron withdrawing or electron donating group, or R10 and R12 taken together with the nitrogen atom to which they are attached and the carbon atom attached to the nitrogen atoms form a 5 or 6 membered nitrogen containing heterocyclic containing 3 or 4 ring carbon atoms, respectively or R10and R11 taken together with the nitrogen atom to which they are attached or R12 and R13 taken together with the nitrogen atom to which they are attached form a 5 or 6-membered heterocyclic ring containing up to 5 ring carbon atoms respectively or R14 and R15 taken together with the nitrogen atom to which they are attached form a 5 or 6-membered heterocyclic ring containing 4 or 5 ring carbon atoms, respectively or R14 taken together with the nitrogen to which it is attached and R16 taken together with the carbon atom to which it is attached form a 5 or 6 membered nitrogen containing heterocyclic ring, containing 4 or 5 ring carbon atoms, respectively;
E is N or CR; and
R is hydrogen or lower alkyl.
Another embodiment of the present invention is directed to an isolated product of Formula I or II.
In a preferred embodiment the compound of Formula I or II is substantially pure.
The present invention is also directed to a process of preparing an amide, including a peptide, which comprises reacting an amine with a carboxylic acid in the presence of an amide forming effective amount of a compound of Formula I or II, and optionally in the presence of a dehydrating reagent.